Identification of a JAK2 mutation involved in vaquez polyglobulia

ABSTRACT

The present invention concerns the V617F variant of the protein-tyrosine kinase JAK2, said variant being responsible for Vaquez Polyglobulia. The invention also relates to a first intention diagnostic method for erythrocytosis and thrombocytosis allowing their association with myeloproliferative disorders, or to the detection of the JAK2 V617F variant in myeloproliferative disorders allowing their reclassification in a new nosological group.

This application is a divisional of U.S. patent application Ser. No.12/234,616, filed on Sep. 19, 2008, which is a continuation of U.S.patent application Ser. No. 11/934,359, filed on Nov. 2, 2007, now U.S.Pat. No. 7,429,456, which is a continuation of U.S. patent applicationSer. No. 10/580,458, filed on May 24, 2006, now U.S. Pat. 7,781,199,which is a U.S. National Stage application of PCT/EP2005/055586, filedOct. 26, 2005, which claims priority to French Patent Application No.0411480, filed Oct. 27, 2004, all of which are incorporated herein byreference in their entirety.

The present invention concerns the V617F variant of the protein-tyrosinekinase JAK2, said variant being responsible for Vaquez Polyglobulia. Theinvention also relates to a first intention diagnostic method forerythrocytosis and thrombocytosis allowing their association withmyeloproliferative disorders, or to the detection of the JAK2 V617Fvariant in myeloproliferative disorders allowing their reclassificationin a new nosological group, and to the identification of specificinhibitors and siRNA.

Vaquez polyglobulia (Polycythemia Vera or PV) is a chronicmyeloproliferative disorder associating true polyglobulia and, often,thrombocytosis and hyperleukocytosis. It is a clonal, acquired diseaseof the hematopoietic stem cell. The hematopoietic progenitors of PV areable to form erythroblast colonies in the absence of erythropoietin(Epo), called “spontaneous colonies”. Hypersensitivity of PVerythroblast progenitors to several other growth factors has also beenshown: Interleukin-3 (IL-3), Granulocyte Macrophage-Stimulating Factor(GM-CSF), Stem Cell Factor (SCF) and Insulin Like Growth Factor (IGF-1).Several teams have taken an interest in the physiopathology of PV, butthe molecular anomaly at the root of the disease remains unknown to date(H. Pahl, 2000).

The hypersensitivity of PV progenitors to several cytokines leads toresearching anomalies involving the signal transduction pathways commonto cytokine receptors. The existence of a molecular marker has neverbeen evidenced in PV, but given the similarities between PV and othermyeloproliferative disorders, CML in particular, it appears probablethat molecular mechanisms close to those induced by Ber-Abl areresponsible for the predominant proliferation of the malignant clone andits end differentiation. This hypothesis was recently confirmed in tworare myeloproliferative disorders, the myeloproliferative disordersassociated with a translocation involving the 8p11 chromosome regionwhich induces constitutive activation of the FGF receptor, and thehypereosinophilic disorder in which a cryptic chromosome deletion leadsto a chimeric gene PDGFRα-FIP1L1. In both cases, the molecular anomaliesare the cause of fusion proteins having a constitutive tyrosine kinaseactivity.

In PV, no recurrent cytogenetic anomaly has been found, even if a 20qdeletion is detected in 10 to 15% of patients, and heterozygosity lossat 9p in approximately 30% of cases (Kralovics, 2002). However, theseanomalies are not specific to the disease.

Since PV cells are Epo-independent, research has been undertaken on thepathway of the Epo receptor (R-Epo). Firstly, the receptor is normalboth structurally and functionally (Hess et al, 1994; Le Couedic et al,1996; Means et al, 1989). The SHP-1 phosphatase which dephosphorylatesR-Epo and JAK2 when Epo stimulation ceases, is normally expressed at RNAand protein level (Andersson et al, 1997; Asimakopoulos et al, 1997).Lower downstream in R-Epo signalling, abnormal activation of STATS hasbeen researched in the polynuclear neutrophils (PNN) of patientspresenting with PV but no anomaly has been found. On the other hand,constitutive phosphorylation of STAT3 has been evidenced in PNNs in 4 PVcases out of 14 examined (Roder, 2001). Finally, the expression of theanti-apoptotic protein bcl-xl, a transcriptional target of STATS, hasbeen studied in immunohistochemistry and by flow cytometry (Silva et al,1998). It was shown that bcl-xl is hyperexpressed in PV erythroblasts,in particular at a more mature stage when this protein is normally nolonger expressed.

In Vaquez polyglobulia, the chief diagnostic criteria to date areclinical (PVSG criteria: Pearson, 2001). Biological diagnosis isessentially based on growing cultures of erythroid progenitors in theabsence of Epo (detection of endogenous colonies). On account of thenecessary expertise for its proper conducting and the substantial“technician-time” required, this test is not available in every centre,and is only reliable when conducted by an experienced laboratory. Inaddition, the test requires medullary cells from the patient to obtaingood sensitivity, which can be a tiresome procedure for the patient.

Using subtraction hybridising techniques, a German team has cloned agene hyperexpressed in the PNNs of PV called PRV1 (Polycythemia Rubravera 1) (Temerinac et al, 2000). The PRV-1 protein belongs to thesuperfamily of uPAR surface receptors. The hyperexpression of mRNAencoding PRV-1 in PV polynuclear neutrophils can be easily detected byreal time RT-PCR; and forms a recently discovered marker of the disease,with no physiopathological role. However, recently published studiesshow that it is neither very sensitive nor very specific.

Spivak J L et al, in 2003 (“Chronic myeloproliferative disorders”;Hematology, 2003; 200 24) describes certain PV markers. The mRNAs of theneutrophilic antigen NBI/CD177 are overexpressed in the granulocytes ofPV patients. This marker does not appear to be a reliable means howeverfor detecting PV, some patients not showing this overexpression or thisoverexpression possibly being observed in patients suffering frommyeloproliferative disorders other than Vaquez polyglobulia. Reducedexpression of the thrombopoietin receptor, Mp1, on platelets is alsofound in PV. Although this anomaly is predominant in PV it is found inother myeloproliferative disorders. In addition, it is a test that isdifficult to carry out and can only be performed in specialisedlaboratories.

Therefore, in the state of the art, no method exists which provides areliable diagnosis of PV. In addition, the only available treatments arenot specific. These relate to phlebotomy to maintain hematocrit withinnormal limits, or the use of cytotoxic agents or of IFN.

Under the present invention we have not only discovered a mutation inthe JAK2 gene in approximately 90% of tested patients, but we have alsoevidenced that this mutation is responsible for constitutive activationof this tyrosine kinase and have shown that its inhibition makes itpossible to block the spontaneous proliferation and differentiation ofPV erythroblasts.

JAK2 belongs to the family of Janus Kinases (JAKs) which group togetherseveral intracytoplasmic tyrosine kinases: JAK1, JAK2, JAK3 and TYK2.The JAK proteins are involved in the intracellular signalling ofnumerous membrane receptors which have no intrinsic tyrosine kinaseactivity, like some members of the superfamily of cytokine receptors andin particular the Epo receptor (R-Epo). The JAK2 protein is encoded by agene which comprises 23 exons. The size of the complementary DNA is 3500base pairs and encodes a protein of 1132 amino acids (130 kD) (FIG. 1).Using PCR and sequencing we have identified a clonal, acquired, pointmutation in exon of JAK2 in nearly 90% of patients suffering from PV.The “GTC” 617 codon, normally coding for a Valine (V) is mutated to“TTC” coding for a Phenylalanine (F). This V617F mutation is not foundin the 25 controls or patients suffering from secondary polyglobulia whowere tested. On the other hand, it D is found in 40% of essentialthrombocytaemias and in 50% of myelofibroses, which means that thismutation defines a new myeloproliferative disorder framework in the sameway as Bcr-Abl defined chronic myeloid leukaemia.

To examine whether the variant of the invention, JAK2 V617F, could bedetected with efficacy using instruments given general wide use inhaematology diagnostic laboratories, we analysed 119 samples frompatients suspected of suffering from a myeloproliferative disorder. Wehave shown that JAK2 V617F is efficiently detected by LightCycler® andTaqMan® technologies, these being slightly more sensitive thansequencing. We then estimated the detection value of JAK2 V617F as firstintention diagnostic test in 88 patients with hematocrit levels of over51%, and it was shown that the mutation corresponded to PV diagnosis inaccordance with WHO criteria (R=0.879) and PVSG criteria (R=0.717) witha positive predictive value of 100% in the context of erythrocytosis. Onthe basis of this data, we propose that the detection of JAK2 V617F ingranulocytes should be considered as a first intention diagnostic testin patients with erythrocytosis, thereby avoiding the measurement of redcell mass, bone marrow procedure and in vitro analysis of the formationof endogenous erythroid colonies. This detection could also be extendedin first intention to all myeloproliferative disorders or theirsuspected presence. This detection will be of particular importance forchronic thrombocytoses for which no certain biological tests exist toconfirm a myeloproliferative disorder. It will also be an important testin the diagnosis of myelofibrosis and for clinical pictures associatedwith thromboses of undetermined aetiology.

Therefore, for the first time, the invention provides a diagnostic tooland opens the way to targeted treatment of PV and of myeloproliferativedisorders associated with this mutation. More specifically, we proposethe detection of the JAK2 V617F mutation as a first intention diagnostictest for erythrocytosis, making it possible to avoid quantification ofred cell mass and erythroid endogenous colonies (EEC) as well as bonemarrow testing in the majority of patients and in chronic thrombocytosisthereby avoiding lengthy aetiological search.

DESCRIPTION OF THE INVENTION

Therefore, according to a first characteristic, the present inventionconcerns the isolated protein JAK 2 (Janus kinase 2), in particular theHomo sapiens Janus kinase 2 protein (NCBI, accession numberNM_(—)004972; G1:13325062) comprising a mutation on amino acid 617(codon 617 of the cDNA starting from ATG) more particularly the V617Fmutation, hereinafter called variant JAK2 V617F such as presented in SEQID No 1 below:

(V617F Homo sapiens Janus kinase 2 or JAK2 V617F) SEQ ID N^(o) 1MGMACLTMTEMEGTSTSSIYQNGDISGNANSMKQIDPVLQVYLYHSLGKSEADYLTFPSGEYVAEEICIAASKACGITPVYFINMFALMSETERIWYPPNHVFHIDESTRHNVLYRIRFYFPRWYCSGSNRAYRHGISRGAEAPLLDDFVMSYLFAQWRHDFVHGWIKVPVTHETQEECLGMAVLDMMRIAKENDQTPLAIYNSISYKTFLPKCIRAKIQDYHILTRKRIRYRFRRFIQQFSQCKATARNLKLKYLINLETLQSAFYTEKTEVKEPGSGPSGEEIFATIIITGNGGIQWSRGKHKESETLTEQDLQLYCDFPNIIDVSIKQANQEGSNESRVVTIHKQDGKNLEIELSSLREALSFVSLIDGYYRLTADAHHYLCKEVAPPAVLENIQSNCHGPISMDFAISKIKKAGNQTGLYVLRCSPKDFNKYFLTFAVERENVIEYKHCLITKNENEEYNLSGTKKNFSSLKDLLNCYQMETVRSDNIIFQFTKCCPPKPKDKSNLLVFRTNGVSDVPTSPTLQRPTHMNQMVFHKIRNEDLIFNESLGQGTFTKIFKGVRREVGDYGQLHETEVLLKVLDKAHRNYSESFFEAASMMSKLSHKHLVLNYGVC F ⁶¹⁷ CGDENILVQEFVKFGSLDTYLKKNKNCINILWKLEVAKQLAWAMHFLEENTLIHGNVCAKNILLIREEDRKTGNPPFIKLSDPGISITVLPKDILQERIPWVPPECIENPKNLNLATDKWSFGTTLWEICSGGDKPLSALDSQRKLQFYEDRHQLPAPKWAELANLINNCMDYEPDFRPSFRAIIRDLNSLFTPDYELLTENDMLPNMRIGALGFSGAFEDRDPTQFEERHLKFLQQLGKGNFGSVEMCRYDPLQDNTGEVVAVKKLQHSTEEHLRDFEREIEILKSLQHDNIVKYKGVCYSAGRRNLKLIMEYLPYGSLRDYLQKHKERIDHIKLLQYTSQICKGMEYLGTKRYIHRDLATRNILVENENRVKIGDFGLTKVLPQDKEYYKVKEPGESPIFWYAPESLTESKFSVASDVWSFGVVLYELFTYIEKSKSPPAEFMRMIGNDKQGQMIVFHLIELLKNNGRLPRPDGCPDEIYMIMTECWNNNVNQRPSFRDLALRVD QIRDNMAG

The invention also concerns equivalents of this protein mutated atposition 617 in other mammals, for example JAK2 V617F in rat (NM031514), porcine, murine (NM-008413) mammals . . . and variants of SEQID No 1 which also comprise one or more alterations which do not affectthe activity and 3D structure of the variant.

The invention also relates to a nucleotide sequence encoding SEQ ID No1, preferably SEQ ID No 2 (sequence of the human JAK2 gene with the TTCcodon instead of GTC on codon 617 (g/t mutation at position 1849hereinafter called G1849T, starting from the ATG marking the start oftranslation).

This sequence may be found in a viral or plasmid vector, or a naked DNAunder the control of a efficient promoter in mammalian cells. Theinvention therefore extends to a vector expressing the JAK2 V617Fprotein.

The vector of the invention may be a cloning and/or expression vectorand may be used to transfect a host cell, in particular a mammaliancell, preferably a human CD34+ progenitor cell.

Model Transgenic Animal of PV and Other Myeloproliferative Disorders

The invention also concerns a non-human transgenic animal expressingrecombinant JAK2 V617F. This animal may preferably be a mouse or rat.Transgenic rats or mice which can be used as models may be obtained byany method commonly used by those skilled in the art, in particular by aKnock-in method (targeted insertion of a sequence) by homologousrecombination or directed recombination with the Cre-LoxP or FLP-FRTsystems in ES cells. According to one preferred embodiment of theinvention, the inventive transgenic cell is obtained by gene targetingof the JAK2 G1849T variant at one or more sequences of the host cellgenome. More precisely, the transgene is inserted stable fashion byhomologous recombination at the homologous sequences in the genome ofthe host cell. When it is desired to obtain a transgenic cell with aview to producing a transgenic animal, the host cell is preferably anembryonic stem cell (ES cell) (Thompson et al, 1989). Gene targeting isthe directed modification of a chromosome locus by homologousrecombination with an exogenous DNA sequence having sequence homologywith the targeted endogenous sequence. There are different types of genetargeting. Here, gene targeting may be used more particularly to replacethe wild-type JAK2 gene by the gene variant JAK2 G1849T or any othergenetically similar variant. In this case, the gene targeting is called“Knock-in” (K-in). Alternatively, gene targeting may be used to reduceor annihilate expression of wild-type JAK2 to insert the gene of theJAK2 variant. This is then called “Knock-out” gene targeting (KO) (seeBolkey et al, 1989). The cell of the invention is characterized in thatthe transgene is integrated stably into the genome of said cell, and inthat its expression is controlled by the regulatory elements of theendogenous gene. By stable integration is meant the insertion of thetransgene into the genomic DNA of the inventive cell. The transgene soinserted is then transmitted to cell progeny. Integration of thetransgene is made upstream, downstream or in the centre of the targetJAK2 endogenous gene. Optionally, one or more positive or negativeselection genes may be used. It is also possible to use DNA homologyregions with the target locus, preferably a total of two, located eitherside of the reporter gene portion or either side of the completesequence to be inserted. By “DNA homology regions” is meant two DNAsequences which, after optimal alignment and after comparison, areidentical for usually at least approximately 90% to 95% of thenucleotides and preferably at least 98 to 99.5% of the nucleotides.Optimal alignment of the sequences for comparison may be made using theSmith-Waterman local homology algorithm (1981), the Neddleman-Wunschlocal homology algorithm (1970), the similarity search method of Pearsonand Lipman (1988), or computer software using these algorithms (GAP,BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,W1). Although as few as 14 bp with 100% homology are sufficient toachieve homologous recombination in bacteria and mammalian cells, longerportions of homologous sequences are preferred (in general the size ofthese portions is at least 2000 bp, preferably at least 5000 bp for eachportion of homologous sequence. Advantageously, the JAK variant sequenceis inserted in the group of elements ensuring endogenous typeregulation, i.e. a group comprising at least the promoter, regulatorsequences (enhancers, silencers, insulators) and the terminating signalsof the endogenous JAK gene.

According to one particular embodiment, the transgene JAK G1849Tcomprises at least the encoding sequence, a positive selection cassettewhether flanked or not by sites specific to the action of therecombinases, e.g. a Lox/Neo-TK/Lox cassette or lox/Neo/lox orFRT/Neo-TK/FRT ou FRT/Neo/FRT cassette possibly also being present atposition 5′ of said sequence, and characterized in that a negativeselection cassette for example containing the DTA and/or TK gene orgenes is at least present at one of the ends of the transgene. Thetransgene of the present invention is preferably directly derived froman exogenous DNA sequence naturally present in an animal cell. This DNAsequence in native form may be altered for example through the insertionof restriction sites needed for cloning and/or through the insertion ofsite-specific recombination sites (lox and flp sequences).

For this purpose, the JAK2 G1849T variant can be cloned in a cloningvector ensuring its propagation in a host cell, and/or optionally in anexpression vector to ensure expression of the transgene. The recombinantDNA technologies used to construct the cloning and/or expression vectorof the invention are known to those skilled in the art. Standardtechniques are used for cloning, DNA isolation, amplification andpurification; enzymatic reactions involving DNA ligase, DNA polymerase,restriction endonucleases are performed following the manufacturer'sinstructions. These techniques, and others are generally conducted inaccordance with Sambrook et al, 1989. The vectors include plasmids,cosmids, bacteriophages, retroviruses and other animal viruses,artificial chromosomes such as YAC, BAC, HAC and other similar vectors.

The methods for generating transgenic cells of, the invention aredescribed in Gordon et al, 1989. Various techniques for transfectingmammalian cells were reviewed by Keon et al., 1990. The inventivetransgene, optionally contained in a linearised or non-linearisedvector, or in the form of a vector fragment, can be inserted in the hostcell using standard methods such as microinjection into the nucleus forexample (U.S. Pat. No. 4,873,191), transfection by calcium phosphateprecipitation, lipofection, electroporation (Lo, 1983), heat shock,transformation with cationic polymers (PEG, polybrene, DEAE-Dextran.) orviral infection (Van der Putten et al, 1985).

When the cells have been transformed by the transgene, they may becultured in vitro or else used to produce non-human transgenic animals.After transformation, the cells are seeded on a nutritional layer and/orin a suitable medium. The cells containing the construct can be detectedusing a selective medium. After a sufficient time to allow the coloniesto grow, they are then collected and analysed to determine whether ornot a homologous recombinant event and/or integration of the constructhas occurred. To screen the clones possibly fulfilling homologousrecombination, positive and negative markers, also called selectiongenes, may be inserted in the homologous recombination vector. Differentsystems for selecting cells producing the homologous recombination eventhave been described (for review U.S. Pat. No. 5,627,059). Said positiveselection gene of the invention is preferably chosen from amongantibiotic-resistant genes. Among the antibiotics a non-exhaustive listcomprises neomycin, tetracycline, ampicilline kanamycin, phleomycin,bleomycin, hygromycin, chloramphenicol, carbenicilline, geneticine,puromycin. The resistance genes corresponding to these antibiotics areknown to those skilled in the art; as an example the resistance gene toneomycin makes the cells resistant to the presence of the G418antibiotic in the culture medium. The positive selection gene may alsobe chosen from among the HisD gene, the corresponding selective agentbeing histidinol. The positive selection gene may also be chosen fromamong the gene of guanine-phosphoribosyl-transferase (GpT), thecorresponding selective agent being xanthine. The positive selectiongene may also be chosen from among thehypoxanthine-phosphoribosyl-transferase gene(HPRT), the correspondingselective agent being hypoxanthine. The selection marker or markers usedto allow identification of homologous recombination events maysubsequently affect gene expression, and may be removed if necessaryusing specific site recombinases such as the Cre recombinase specific toLox sites (Sauer, 1994; Rajewsky et al, 1996; Sauer, 1998) or FLPspecific to FRT sites (Kilby et al, 1993).

The positive colonies, i.e. containing cells in which at least onehomologous recombinant event has occurred, are identified by Southernblotting analysis and/or PCR techniques. The expression level, in theisolated cells or cells of the inventive transgenic animal, of the mRNAcorresponding to the transgene may also be determined by techniquesincluding Northern blotting analysis, in situ hybridisation analysis, byRT-PCR. Also, the cells or animal tissues expressing the transgene maybe identified using an antibody directed against the reporter protein.The positive cells may then be used to conduct embryo handlingprocedures in particular the injection of cells modified by homologousrecombination into the blastocysts.

Regarding mice, the blastocysts are obtained from 4 to 6-weeksuperovulated females. The cells are trypsinated and the modified cellsare injected into the blastocele of a blastocyst. After injection, theblastocysts are inserted into the uterine horn of pseudo-pregnantfemales. The females are then allowed to reach full term and theresulting offspring are analysed to determine the presence of mutantcells containing the construct. Analysis of a different phenotypebetween the cells of the newborn embryo and the cells of the blastocystor ES cells makes it possible to detect chimeric newborn. The chimericembryos are then raised to adult age. The chimera or chimeric animalsare animals in which only a sub-population of cells contains an alteredgenome. Chimeric animals having the modified gene or genes are generallycross-bred between each other or with a wild-type animal to obtaineither heterozygous or homozygous offspring. Male and femaleheterozygotes are then cross-bred to generate homozygous animals. Unlessotherwise indicated, the non-human transgenic animal of the inventioncomprises stable changes in the nucleotide sequence of germ line cells.

According to another embodiment of the invention, the inventivenon-human transgenic cell may be used as nucleus donor cell for thetransfer of a nucleus, or nuclear transfer. By nuclear transfer is meantthe transfer of a nucleus from a living donor cell of a vertebrate, anadult or foetal organism, into the cytoplasm of an enucleated receivercell of the same species or a different species. The transferred nucleusis reprogrammed to direct the development of cloned embryos which canthen be transferred to foster females to produce the foetuses andnewborn, or can be used to produce cells of the inner cell mass inculture. Different nuclear cloning techniques may be used; among these,non-exhaustive mention may be made of those subject of patentapplications WO 17500, WO 97/07668, WO 97 07669, WO 98 30683, WO 9901163 and WO 99 37143.

Therefore, the invention also extends to a non-human transgenic animalcomprising a recombinant sequence encoding JAK2 V617F. These animals maybe homozygous or heterozygous (JAK2 V617F/JAK V617F or JAK2 V617F/JAK2).In particular, these animals reproduce Vaquez polyglobulia but also anymyeloproliferative disorder induced by JAK2 V617F. They can therefore beused to conduct functional screening of tyrosine kinase inhibitors,especially screening of inhibitors specific to JAK2 V617F.

Another alternative consists of injecting a viral vector (retrovirus orlentivirus or others) able to express the JAK2 V617F variant inhematopoietic stem cells, progenitor cells or ES cells also with a viewto producing models of Vaquez Polyglobulia or other myeloproliferativedisorders.

Diagnostic tools

According to a third characteristic, the invention relates to diagnostictools with which to detect the presence or absence of the JAK2 V617Fmutation in mammals suffering from or likely to show amyeloproliferative disorder, in particular in patients presenting withpolyglobulia and who are suspected of having symptoms of Vaquezpolyglobulia, thrombocytaemia and/or myelofibrosis.

In this respect, the invention relates to primers and probes with whichto detect the presence or absence of the mutation in the SEQ ID No 2sequence described above. More particularly, the invention pertains toan isolated nucleic acid having a sequence of at least 10, 12, 15, 20,30, 40 or 50 consecutive nucleotides (e.g. 10 to 30 nucleotides or 10 to25 nucleotides) of exon 12 or of the sequence SEQ ID No 3 or No 4 belowand including the mutated t¹⁸⁴⁹ nucleotide, of 10 to 30 nucleotides forexample.

SEQ ID N^(o) 3ctcatatgaaccaaatggtgtttcacaaaatcagaaatgaagatttgatatttaatgaaagccttggccaaggcacttttacaaagatttttaaaggcgtacgaagagaagtaggagactacggtcaactgcatgaaacagaagttcttttaaaagttctggataaagcacacagaaactattcagagtctttctttgaagcagcaagtatgatgagcaagctttctcacaagcatttggttttaaattatggagtatgtt ¹⁸⁴⁹tctgtggagacgagaatattctggttcaggagtttgtaaaatttggatcactagatacatatctgaaaaagaataaaaattgtataaatatattatggaaacttgaagttgctaaacagttggcatgggccatgcattttctagaagaaaacacccttattcatgggaatgtatgtgccaaaaatattctgcttatcagagaagaagacaggaagacaggaaatcctcctttcatcaaacttagtgatcctggcattagtattacagttttgccaaaggacattcttcaggag

The underlined sequence designates an example of upstream or downstreamareas allowing the design of probes or primers specific to the mutationat position 1849 (SEQ ID No 4).

EXAMPLE OF DIFFERENT PREFERRED PRIMERS AND PROBES OF THE INVENTION

On DNA, PCR PRIMERS: JAK2EXON12-PCRF SENSE (SEQ ID N^(o) 5)5′-GGGTTTCCTCAGAACGTTGA-3′ (54804-54823) JAK2EXON12-PCRR ANTI-SENSE(SEQ ID N^(o) 6) 5′-TTGCTTTCCTTTTTCACAAGA-3′ (55240-55260)ON DNA, SEQUENCING PRIMERS: JAK2EXON12SEQF SENSE (SEQ ID N^(o) 7)5′-CAGAACGTTGATGGCAGTTG-3′ (54813-54832) JAK2EXON12SEQR ANTI-SENSE(SEQ ID N^(o) 8) 5′-TGAATAGTCCTACAGTGTTTTCAGTTT-3′ (55207-55233)ON cDNA, PCR AND SEQUENCING PRIMERS SENSE (SEQ ID N^(o) 9)5′-CAACCTCAGTGGGACAAAGAA-3′ (1386-1407) ANTI-SENSE (SEQ ID N^(o) 10)5′-GCAGAATATTTTTGGCACATACA-3′ (2019-2041)SNPS PROBES AND DETECTION OF MUTATION AND siRNA (1829-1870):(SEQ ID N^(o) 11) TTTTAAATTATGGAGTATGTGTCTGTGGAGACGAGAATATTCGENOTYPING on LightCycler (DNA of PNN or marrow): Oligo “S” (sense)(SEQ ID N^(o) 15) GGCAGAGAGAATTTTCTGAAC Oligo “R” (anti-sense)(SEQ ID N^(o) 16) GCTTTCCTTTTTCACAAGATA Sensor wt (SEQ ID N^(o) 17)GTCTCCACAGACACATACTCCATAA 3′FL Anchor JAK2 (SEQ ID N^(o) 18)5′-LC Red640AAAACCAAATGCTTGTGAGAAAGCT 3′-PHGENOTYPING on LightCycler (e.g. cDNA of platelets) cJAK2F(SEQ ID N^(o) 19) GCACACAGAAACTATTCAGAGTC cJAK2S (SEQ ID N^(o) 20)AGCAGCAAGTATGATGAGC cJAK2A (SEQ ID N^(o) 21) CTAGTGATCCAAATTTTACAAACTcJAK2R (SEQ ID N^(o) 22) GTTTAGCAACTTCAAGTTTCC Sensor wt(SEQ ID N^(o) 23) GTCTCCACAGACACATACTCCATAA3′-FL Anchor JAK2(SEQ ID N^(o) 24) 5′-LC Red640AAAACCAAATGCTTGTGAGAAAGCT3′-PHGENOTYPING using TagMan technology (e.g. on DNAof Bone Marrow mononuclear cells).Recognition using fluorescent probes specific toallele and single strand DNA. PCR reaction Primer sequence sense:(SEQ ID N^(o) 25) AAGCTTTCTCACAAGCATTTGGTTT Primer sequence anti-sense:(SEQ ID N^(o) 26) AGAAAGGCATTAGAAAGCCTGTAGTT Reporter 1 Sequence (VIC):(SEQ ID N^(o) 27) TCTCCACAGACACATAC Reporter 2 Sequence (FAM):(SEQ ID N^(o) 28) TCCACAGAAACATAC.

According to further characteristic, the invention relates to an invitro or ax vivo diagnostic method with which to detect the presence orabsence of the JAK2 V617F mutation in a sample.

Tests With the Nucleic Acids of the Invention

Under a first embodiment, the G1849T variant (corresponding to the JAK2V617F mutation) can be detected by analysis of the nucleic acid moleculeof the JAK2 gene. Within the scope of the present invention, by “nucleicacid” is meant mRNA, genomic DNA or cDNA derived from mRNA.

The presence of absence of the nucleic acid of the G1849T variant can bedetected by sequencing, amplification and/or hybridisation with aspecific probe and specific primers such as described above: sequencederived from SEQ ID No 3 or 4 and SEQ ID No 5 to 11, or further SEQ IDNo 15 to 24.

The invention therefore proposes an ex vivo or in vitro method todetermine the presence of absence of the G1849T variant of the JAK2 genein a sample taken from a patient suffering from PV or likely to developPV or any other myeloproliferative disorder, in particularerythrocytosis, thrombocytaemia and myelofibrosis disorders, the methodcomprising:

-   -   a) obtaining a nucleic acid sample from the patient,    -   b) detecting the presence or absence of the G1849T variant of        the JAK2 gene in said nucleic acid sample.        characterized in that the presence of the G1849T variant is an        indication of PV or any other myeloproliferative disorder.

The nucleic acid sample may be obtained from any cell source or tissuebiopsy. These cells must be of hematopoietic origin and may be obtainedfrom circulating blood, from hematopoietic tissue or any fluidcontaminated with blood cells. The DNA can be extracted using any knownmethod in the state of the art such as the methods described in Sambrooket al (1989). The RNA can also be isolated, for example from tissuesobtained during a biopsy, using standard methods well known to thoseskilled in the art, such as extraction byguanidiumthiophenate-phenol-chloroform.

The G1849T variant of the JAK2 gene can be detected in a RNA or DNAsample, preferably after amplification For example, the isolated RNA canbe subjected to reverse transcription followed by amplification, such asa RT-PCR reaction using oligonucleotides specific to the mutated site orwhich allow amplification of the region containing the mutation, forexample exon 12 or sequence SEQ ID No 3 or 4. The expression“oligonucleotide” is used here to designate a nucleic acid of at least10, preferably between 15 and 25 nucleotides, preferably less than 100nucleotides, and which is able to hybridise to the genomic DNA of JAK2,to cDNA or to mRNA.

The oligonucleotides of the invention may be labelled using anytechnique known to those skilled in the art, such as radioactive,fluorescent or enzymatic labellers. A labelled oligonucleotide can beused as a probe to detect the presence or absence of the G1849T variantof the JAK2 gene.

Therefore, the probes and primers of use in the invention are thosewhich hybridise specifically to the region of the JAK2 gene in thevicinity of the nucleotide at position 1849 (numbering as from the ATGmarking the start of transcription).

In the above-explained method, the nucleic acids may be PCR amplifiedbefore detection of the allelic variation. The methods for detecting theallelic variation are described for example in “Molecular Cloning—ALaboratory Manual” Second Edition, Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory, 1989) and Laboratory Protocols for MutationDetection, Ed. U. Landegren, Oxford University Press, 1996, and PCR2^(nd) edition by Newton & Graham, BIOS Scientific Publishers Limited1997.

In this respect it is possible to combine an amplification step followedby a detection step allowing discrimination between the samples inrelation to the presence or absence of the sought variant.

Different techniques adapted for this purpose are described in EP 1 186672 such as DNA sequencing, sequencing by SSCP, DGGE, TGGEhybridisation, heteroduplex analysis, CMC, enzymatic mismatch cleavage,hybridisation based solid phase hybridisation, DNA chips, Taqman™hybridisation phase solution (U.S. Pat. Nos. 5,210,015 and 5,487,972)and the RFLP technique.

Detection can be conducted using different alternative methods: FRET,fluorescence quenching, polarised fluorescence, chemiluminescence,electro-chemiluminescence, radioactivity and colorimetry.

The method of the invention can include or exclude the steps consistingof obtaining the sample and extracting the nucleic acid from saidsample.

As indicated above the sample used may be blood or any other body fluidor tissue obtained from an individual. After the nucleic acid extractionand purification steps, PCR amplification using the above-describedprimers can be used to improve signal detection.

Therefore, the method of the invention may comprise the amplificationstep with said primers, followed by a hybridisation step with at leastone probe, preferably two probes which hybridise under conditions ofhigh stringency to the sequences corresponding to the region of theG1849T mutation described above, and detection of the signal produced bythe labellers of said probes.

For example, the invention particularly concerns an in vitro method todetermine the presence or absence of the G1849T variant of the JAK2 genein the sample of a patient with PV or likely to develop PV or any othermyeloproliferative disorder, comprising the detection of the presence orabsence of the G1849T variant of the JAK2 gene in said nucleic acidsample by means of one or SNPs (Single Nucleotide Polymorphism) specificto the G1849T mutation of the JAK2 gene, in particular SEQ ID No 17, 18or 23 and 24. characterized in that the presence of the G1849T variantis an indication of PV or of any other myeloproliferative disorder.

This detection by means of SNPs may be implemented using. Taqman®Technology enabling allelic discrimination. Essentially, this methodconsists of the recognition, by the fluorescent probes specific toallele 1849, of JAK2 on single strand DNA and comprises a PCR reaction(with a polymerase with 5′ exonuclease activity), detection offluorescence emission specific to the allele of the hybridised SNPs,determination of the genotype by reading end point fluorescence(obtaining an image showing clusters of mutated homozygous, heterozygousand normal DNA).

Detection of the Mutated Protein JAK2 V617F

According to another embodiment, the variant can be detected-directlywithin the JAK2 protein.

For this purpose, the invention concerns an ex vivo or in vitro methodfor detecting the presence or absence of the JAK2 V617F variant in asample from a patient suffering from or likely to develop PV or anyother myeloproliferative disorder, in particular erythrocytosis,thrombocytaemia and myelofibrosis, method consisting of:

-   a) obtaining a sample from the patient,-   b) detecting the presence or absence of the JAK2 V617F variant,    characterized in that the presence of said variant is an indication    of PV or of any other myeloproliferative disorder.

Said JAK V617F variant can be detected by any suitable method known inthe state of the art.

More particularly, a sample taken from an individual can be contactedwith an antibody specific to the V617F variant of the JAK2 protein, e.g.an antibody which is able to distinguish between the V617F variant andthe non-mutated JAK2 protein (and any other protein).

The antibodies of the present invention can be monoclonal ;5 orpolyclonal antibodies, single chain or double chain, chimeric orhumanised antibodies or portions of immunoglobulin molecules containingthe portions known in the state of the art to correspond to the antigenbinding fragments [human fragment, human F(ab′)2 and F(v)].

These antibodies may be immunoconjugated, for example with a toxin or amarker.

The protocols for obtaining polyclonal antibodies are well known tothose skilled in the art. Typically, said antibodies can be obtained byadministering the JAK2 V617F variant via subcutaneous injection intowhite New Zealand rabbits previously prepared to obtain a pre-immunityserum. The antigens can be injected up to 100 μl per site at 6 differentsites. The rabbits are prepared two weeks before the first injection,then periodically stimulated with the same antigen approximately threetimes every six weeks. A serum sample is then obtained ten days aftereach injection. The polyclonal antibodies are then purified of the serumby affinity chromatography using the JAK2 V617F protein to capture theantibodies.

Monoclonal antibodies are preferred to polyclonal antibodies on accountof their high specificity.

Obtaining said monoclonal antibodies is within the reach of personsskilled in the art bearing in mind that the JAK2 V617F variant has adifferent 3D structure to the wild-type JAK2 protein. The expression“monoclonal antibody” means an antibody which is able to recognize onlyan epitope of an antigen.

Monoclonal antibodies can be prepared by immunizing a mammal, e.g. amouse, rat or other mammals with the purified JAK2 V617F variant. Thecells of the immunised mammal producing the antibodies are isolated andfused with the cells of myelomas or hetero-myelomas to produce hybridcells (hybridomas).

The hybridoma cells producing the monoclonal antibody are used asproduction source for the antibody. The techniques for generatingantibodies which do not involve immunisation are also concerned by theinvention. For example “phage display” technology.

The antibodies directed against the JAK2 V617F variant may in some casesshow a cross reaction with the wild-type JAK2 protein. If this is thecase, a selection of the antibodies specific to the V617F variant isrequired. In this respect affinity chromatography may be used forexample with the wild-type JAK2 protein to capture the antibodiesshowing a cross reaction with wild-type JAK2.

Therefore, the invention relates to a monoclonal antibody specificallyrecognizing the JAK2 V617F variant and to the hybridoma lines producingthe antibody.

The invention also concerns an ELISA test using said antibody to detectthe presence or absence of the JAK2 V617F variant in a sample.

One alternative to the use of antibodies may for example consist ofpreparing and identifying haptamers which are classes of moleculesenabling specific molecular recognition.

Haptamers are oligonucleotides or oligopeptides which can virtuallyrecognize any class of targeted molecules with high affinity andspecificity.

Kits

According to another characteristic, the invention relates to kits todetermine whether a patient is suffering from Vaquez polyglobulia oranother myeloproliferative disorder involving the JAK2 V617F variant.

The inventive kit may contain one or more probes or primers such asdefined above for the specific detection of the presence or absence ofthe G1849T mutation in the JAK2 gene.

The kit may also contain a heat-resistant polymerase for PCRamplification, one or more solutions for amplification and/or thehybridisation step, and any reagent with which to detect the marker.

According to another embodiment, the kit contains an antibody such asdefined above.

The kits of the invention may also contain any reagent adapted forhybridisation or immunological reaction on a solid carrier.

The method and the detection kit are advantageously used for thesub-population of patients showing a hematocrit level higher than 51%.The method and the detection kit are also advantageously used for thesub-population of patients showing a platelet count of more than 450000.

siRNA of the Invention

According to a fourth characteristic, the invention also relates tosiRNAs enabling a reduction of more than 50%, 75%, 90%, 95% or more than99% in the expression of JAK2 mutated at position 617, in particularJAK2 V617F. These siRNAs can be injected into the cells or tissues bylipofection, transduction or electroporation. They can be used tospecifically destroy the mRNAs encoding JAK2 V617F thereby entailingnumerous possible therapeutic applications, in particular the treatmentof Vaquez polyglobulia.

srRNAs are described in U.S. 60/068,562 (CARNEGIE). The RNA ischaracterized in that it has a region with a double strand structure(ds). Inhibition is specific to the target sequence, the nucleotidesequence of one strand of the RNA ds region comprising at least 25 basesand being identical to the portion of the target gene. The nucleotidesequence of the other strand of the RNA ds region is complementary tothat of the first strand and to the portion of the target gene. Also,application WO 02/44 321 (MIT/MAX PLANCK INSTITUTE) describes a doublestrand RNA (or oligonucleotides of same type, chemically synthesized) ofwhich each strand has a length of 19 to 25 nucleotides and is capable ofspecifically inhibiting the post-transcriptional expression of a targetgene via an RNA interference process in order to determine the functionof a gene and to modulate this function in a cell or body. Finally, WO00/44895 (BIOPHARMA) concerns a method for inhibiting the expression ofa given target gene in a eukaryote cell in vitro, in which a dsRNAformed of two separate single strand RNAs is inserted into the cell, onestrand of the dsRNA having a region complementary to the target gene,characterized in that the complementary region has at least 25successive pairs of nucleotides. Persons skilled in the art may refer tothe teaching contained in these documents to prepare the siRNAs of theinvention.

More specifically, the invention relates to double strand RNAs ofapproximately 15 to 30 nucleotides, or preferably around 19 nucleotidesin length that are complementary (strand 1) and identical (strand 2) tosequence SEQ ID No 3 comprising the G1849T mutation. These siRNAs of theinvention may also comprise a dinucleotide TT or UU at the 3′ end.

Numerous programmes are available for the design of the siRNAs of theinvention:

-   -   “siSearch Program”        -   (Improved and automated prediction of effective siRNA″,            Chaml A M, Wahlesdelt C and Sonnhammer E L L, Biochemical            and Biophysical research Communications, 2004).    -   “SiDirect”        -   (Direct: highly effective, target-specific siRNA design            software for mammalian RNA interference, Yuki Naito et al.            Nucleic Acids Res, Vol. 32, N2 Web Server Issue© Oxford            University Press, 2004).    -   “siRNA Target Finder” by Ambion.    -   “siRNA design tool” by Whitehead Institute of Biomedical        research at the MIT. Other programmes are available.

For example, for the sequence TATGGAGTATGTT¹⁸⁴⁹TCTGTGGAGA (SEQ ID No 12)the sense siRNA is UGGAGUAUGUUUCUGUGGAdTdT (SEQ ID No 13) and theanti-sense siRNA is UCCACAGAAACAUACUCCAdTdT (SEQ ID No 14).

In one particular embodiment, the siRNAs of the invention describedabove are tested and selected for their capability of reducing, evenspecifically blocking the expression of JAK2 VI617F, affecting as littleas possible the expression of wild-type JAK2. For example, the inventionconcerns siRNAs allowing a reduction of more than 80%, 90%, 95% or 99%of the expression of JAK2 V617F and no reduction or a reduction of lessthan 50%, 25%, 15%, 10% or 5% or even 1% of wild-type JAK2.

For example, the siRNAs of the invention can be selected from the groupconsisting of:

(SEQ ID N^(o) 29) UGGAGUAUGUUUCUGUGGA (SEQ ID N^(o) 30)GGAGUAUGUUUCUGUGGAG (SEQ ID N^(o) 31) GAGUAUGUUUCUGUGGAGA

According to another embodiment, the invention concerns a ddRNAimolecule such as described generic fashion in application WO 01/70949(Benitec) but specifically targeting JAK2 V617F. The ddRNAi of theinvention allows extinction of the sequence coding for JAK2 V617F andcomprises (i) an identical sequence to SEQ ID No 3, 4 or 11; (ii) asequence complementary to the sequence defined under (i); (iii) anintron separating said sequences (i) and (ii); the introduction of thisconstruct in a cell or tissue producing an RNA capable of altering theexpression of JAK2 V617F.

The invention also relates to a genetically modified non-human animalcomprising one or more cells containing a genetic construct capable ofblocking, delaying or reducing the expression of JAK2 V617F in theanimal. The method for producing said genetically modified animal isdescribed in WO 04/022748 (Benitec).

Screening Methods

According to a fifth characteristic, the subject of the invention is amethod for screening inhibitors specific to JAK2 V617F.

By “specific inhibitors” is meant compounds having a ratio of IC50 onJAK2/IC50 on JAK2 V617F of more than 5, 10, 25, or even 50. For examplethe compound has an IC50 on JAK2 V617F of less than 1 μM, preferably 100nM, whereas it has an IC50 on JAK2 of more than 5 μM or 10 μM.

This method can be implemented using the protein of the invention, amembrane fraction containing said protein, a cell expressing saidprotein or a non-human transgenic animal such as described above.

Therefore, the invention relates to a test with which to determine thespecific inhibition of JAK2 V617F by one or more compounds, comprisingthe steps consisting of contacting one or more compounds with theabove-described JAK2 V617F protein, a membrane fraction containing JAK2V617F or a cell expressing JAK2 V617F as described above underconditions suitable for fixing and detecting the specific fixationand/or inhibition of JAK2 V617F.

This method may also comprise measurement of the fixing onto wild-typeJAK2.

This method may also consist of a succession of tests of severalmolecules and comprise a selection step to select molecules showing anIC50 for JAK V617F of less than 1 μM, preferably 100 nM.

This method may also comprise a negative selection step of theabove-mentioned molecules which have an IC50 for JAK2 of less than 5 μM,or 1 μM.

The invention concerns in vitro screening such as described above inwhich immunoprecipitation is used to determine the inhibitedphosphorylation of JAK2 V617F.

The invention also relates to in vivo screening on CD34-JAK2 V617Fprogenitor cells which are capable of differentiating withouterythropoietin (Epo). Said cells are isolated from patients with Vaquezpolyglobulia. The CD34-JAK2 V617F cells can be placed in culture in amedium containing SCF and IL-3. The compounds are added to the culturemedium and the proliferating capacity of the cells is determined andtheir ability to differentiate into 36+/GPA− cells. The compoundsselected are those for which a decrease in 36+/GPA− clones is observed.Hence, the invention relates to the above screening method using primaryCD34+ JAK V617F progenitor cells which are capable of differentiatingwithout erythropoietin (Epo) or using cell lines which have becomefactor independent through the introduction of the JAK2 V617F variant.The same type of test can be conducted on marrow cultures of CFU-E typein a semi-solid medium with direct testing of the compound regarding theinhibition of spontaneous colony growth.

It is also possible to use any mammalian cell line described aboveexpressing recombinant JAK V617F.

The invention also relates to a method for identifying candidatemedicinal products, comprising the steps consisting of administeringcompounds to a non-human transgenic animal expressing JAK2 V617F such asdescribed above, said animal reproducing Vaquez polyglobulia and/orhaving a myeloproliferative disorder associated with the presence ofJAK2 V617F, of determining the effect of the compound and selectingcandidate medicinal products which are seen to cause a reduction orblocking of proliferation and of spontaneous erythroblastdifferentiation in Vaquez polyglobulia or a reduction in cellproliferation associated with the presence of JAK2 V617F.

More particularly, this method is performed with a JAK2 V617F K-in mouseor JAK2 V617F K-in rat such as described above.

Among these compounds, mention may be made for example of siRNAstargeting the mutated exon 12 of JAK2 as described above, in particularsiRNAs targeting sequence SEQ ID No 3, 4 or sequence SEQ ID No 11comprising the mutated t¹⁸⁴⁹ nucleotide.

A further characteristic of the invention concerns the use of saidabove-described siRNAs or ddRNAi, and compounds specifically inhibitingJAK2 V617F to produce a medicinal product. Said medicinal product isparticularly intended for the treatment of blood cancers, in particularmyeloproliferative disorders including Vaquez polyglobulia, essentialthrombocythaemia, myeloid splenomegaly or primitive myelofibrosis andchronic myeloid leukaemia. Said medicinal product is also intended forthe treatment of other malignant hemopathies, associated with the JAK2V617F mutation, and optionally solid tumours, carcinomas, melanomas andneuroblastomas which express JAK2 V617F.

For the remainder of the description and for the examples reference ismade to the figures whose keys are described below:

KEYS TO FIGURES

FIG. 1: Discovery of the key role of JAK2 in PV

-   -   In the basal state, JAK2 is fixed to box 1 in the        non-phosphorylated state. The binding to Epo alters the        conformation of the receptor and enables transphosphorylation of        JAK2 which in return phosphorylates the intracytoplasmic        residues of Epo-R thereby recruiting the different positive (−>)        or negative (−|) effectors of signal transduction.

FIG. 2: Design of a culture model of PV CD34+ progenitors that areerythropoietin-independent.

-   -   2A—culture with Epo, SCF and IL-3    -   2B—culture without Epo

FIG. 3: Inhibition of JAK-STAT, Pi3-K and Src kinase pathways preventsspontaneous erythroid differentiation.

FIG. 4: Protocol for inhibiting JAK2 in PV progenitors

FIG. 5: Results of JAK2 inhibition in PV progenitors

-   -   5A—Reduction in the cloning capacity of 36+/gpa−. Culture D1-D6        in SCF-IL3, electroporation D5, sorting D6 (morpho/36/gpa−).        -   Methyl SCF alone. Count on D13 (D7 post-sorting).    -   5B—Structure of JAK2 with V617F mutation (exon 12).

FIG. 6: Genotyping analyses of SNPs to detect the JAK2 V617F mutation ingenomic DNA using LightCycler® and TaqMan® technologies

-   -   A and B: Detection of the mutation by the fusion analysis curve        of LightCycler® with FRET hybridisation probes. A: Experiments        with various dilutions of HEL DNA in DNA TF-1 are shown. The        peak JAK2 V617F (57°) is still detectable at a dilution of 1%.        B: Results of representative patient samples (#1: homozygous;        #2: heterozygous; #3: weak; #4: non mutated).    -   C. Detection of the mutation by TaqMan® allele specific        amplification. Experiments with dilution of HEL DNA (HEL 100 to        1%: empty squares; TF-1 cells: empty circles) and a few        representative patient samples are shown (black crosses). #a:        homozygous patients; #b: heterozygous patients; #c: weak        patient; #d: non-mutated patients.

FIG. 7: Proposed diagnostic datasheet to diagnose erythrocytosis (i.e.hematocrit level over 51%).

-   -   The number of patients concerned at each stage of the datasheet        is written next to each item (n), only those patients showing        all clinical data being listed here (n=81). Detection of JAK2        V617F as a first intention diagnostic test would have prevented        58/81 patients from undergoing other investigations to diagnose        a PV type myeloproliferative disorder.

FIGS. 8 and 9: Expression of V617F Jak2 in HEL cells is reduced 24 hoursafter treatment with siRNA specific to JAK2 V617F Jak2(siRNA #1, 3 and4).

-   -   0 to 6: HEL cells treated (1 to 6) or non-treated (0) with siRNA        V617F Jak2.    -   C+: 293HEK cells transfected with the V617F Jak2RV vector    -   C−: 293 HEK

FIG. 10: Level of WT Jak2 expression in K562 cells remains unchanged 24hours after treatment with siRNA specific to Jak2 V617.

-   -   Je: K562 cells treated with siRNA WT Jak2    -   0 to 6: K562 cells treated with siRNA V617F Jak2    -   C−: 293HEK (no expression of JAK2).

EXAMPLE 1 Identification of the JAK2 V617F Mutation in 39/43 Patients

The design of a cell culture model of pathological progenitors and theuse of biochemical inhibitors enabled us to evidence that theJAK2-STAT5, P13 kinase and Src kinase pathways are necessary forEpo-independent differentiation of PV progenitors (Ugo et al 2004).These results reassured our hypothesis that the primary molecular lesioncausing PV must be an anomaly of a key protein leading to deregulationof a signalling pathway, like the mutation of a tyrosine kinaseimparting a constitutive activity. Nonetheless, it is the study of 43patients suffering from Vaquez polyglobulia which made it possible toidentify the key role played by the JAK2 protein which is the proteinlocated the most upstream in these different signalling pathways, andwhich is common to the signalling pathways of cytokine receptors forwhich response anomalies have been described in PV. We examined theinvolvement of the JAK2 protein kinase in the physiopathology of PVtaking three complementary approaches:

-   -   a functional approach (inhibition of JAK2 in PV cells by        interfering RNA)    -   a genomic approach (sequencing of the 23 exons of the gene), and    -   a biochemical approach to search a JAK2 phosphorylation anomaly,        the cause of a constitutive activation.

The biological material used was derived from consenting patientssuffering from polyglobulia and corresponds to residues of samples takenfor diagnostic purposes and sent to the Hotel Dieu Central haematologylaboratory, or to therapeutic phlebotomy.

1.1. Functional Study

Study of the JAK2 function in the erythroblasts of patients with Vaquezpolyglobulia was conducted by using electroporation to transduce the PVerythroblasts with a siRNA specific to the JAK2 sequence (AMBION,Huntingdon, England) recognizing as target a sequence located on exon 15of the mRNA of JAK2. We have shown that the inhibition of JAK2 stronglyreduces the cloning capacity and “spontaneous” differentiation of PVprogenitors in the absence of Epo. Normal erythroblast progenitorstransfected with siRNA JAK2 show a reduction in clonogenic potential of70% compared with the control siRNA, which confirms the efficacy oftransfection with siRNA JAK2. In PV, the effects of JAK2 inhibition inthe erythroblast progenitors were studied in an Epo-free culture model,making it possible to study the cells of the malignant clone. Wecompared the clonogenic potential, apoptosis and differentiation of thePV erythroblast progenitors after transfection with a siRNA JAK2 withrespect to a control siRNA. Study of the clonogenic potential of PVprogenitors cultured without Epo shows a very marked reduction in thenumber of colonies after transfection of siRNA JAK2 compared withcontrol siRNA. Flow cytometry of the apoptosis of these cells shows asignificant increase in the apoptosis of cells transfected with siRNAJAK2 compared with non-relevant siRNA (70 versus 53%). Study of theeffects of siRNA JAK2 on differentiation (acquisition of Glycophorin Adetected by flow cytometry) only shows a slight difference between theprogenitors transfected with siRNA JAK2 versus control siRNA.

The results of the cell studies therefore showed that JAK2 is necessaryfor Epo-independent differentiation of PV erythroid progenitors. Theinitial results of biochemical studies (immunoprecipitation) show moreextended phosphorylation of JAK2 after depriving PV erythroidprogenitors of cytokines, as compared with normal cells.

1.2 Genomic Study of JAK2

PCR on the 23 exons was set up on a normal individual using genomic DNA.We then examined 3 patients suffering from PV after extracting thegenomic DNA from erythroid cells obtained in vitro after cell culture.

We identified a point mutation located in exon 12 of JAK2, present in 2out of the 3 patients tested. This mutation concerns base no 1849 of theencoding sequence ([numbering starting at ATG], GenBank NM_(—)004972)and transforms codon 617 of the JAK2 protein (V617F).

-   -   normal 617 codon: gtc code for a Valine (V)    -   mutated 617 codon: ttc code for a Phenylalanine (F)

Using the databases published on the Internet we were able to verifythat it is not a known polymorphism.

We then widened the cohort. To date the mutation has been found in 39patients with PV out of the 43 cases tested. No control (15 casestested) or patient with secondary polyglobulia (18 cases tested) werefound to carry the mutation.

Sequencing Results in Patients

-   -   39 mutated/43 PV tested (90%)    -   2/3 heterozygotes    -   13/39 “homozygotes” i.e. 30% of cases (same proportion as the        loss of heterozygosity at 9 p).        Controls    -   0 mutated cases out of 33 controls tested:        -   including 15 normal individuals        -   and 18 secondary polyglobulias (no spontaneous colonies).

The discovery of this anomaly of JAK2 accounts for the hypersensitivityto numerous growth factors involved in PV (Epo, TPO, IL-3, IL-6, GM-CSF,insulin). Indeed, JAK2 is a protein involved in the signalling pathwaysof the receptors of these cytokines.

Also, the association of JAK2 with R-Epo is particular in that JAK2 isfixed to E-Epo constitutively: the JAK2/R-Epo association initiated inthe Golgi apparatus is necessary for the processing of R-Epo at themembrane of the erythroblasts. A JAK2 anomaly, the cause ofmodifications to the association of JAK2 with R-Epo, could thereforeaccount for the medullary hyperplasia predominance on the erythroblastline, whereas this protein is involved in numerous signalling pathways.Also, Moliterno et al (Moliterno et al, 1998; Moliterno and Spivak,1999) have evidenced faulty membrane expression of mpl related to aglycosylation defect. It is possible that JAK2, by analogy with R-Epo,is necessary for the processing of c-mp1. The anomaly of JAK2 could thenexplain the lack of membrane expression of c-mp1, found in PV.

JAK2 binds to R-Epo on its proximal domain, at a highly conserveddomain, Box2. In the absence of Epo stimulation, JAK2 is constitutivelyfixed to R-Epo, but in a non-phosphorylated form, hence non-active.After stimulation of the receptor by Epo, the two JAK2 moleculesphosphorylate, and then phosphorylate R-Epo enabling the recruitmentthen the phosphorylation of other proteins involved in signaltransduction, such as the proteins STATS, Grb2, P13K. The JAK2 protein,like all JAKs, has a functional kinase domain (JH1), a pseudo-kinasedomain with no tyrosine kinase activity (JH2), and several conserveddomains (JH3-JH7), characteristic of members of the JAK family. The JH2domain appears to be involved in regulating the tyrosine-kinase activityof JAK2. According to available JAK2 protein mapping data (Lindauer,2001), amino acid 617 is located in this JH2 domain and, followingmodelling studies, in a region of particular importance for theregulation of kinase activity:

Over and above the physiopathological interest of this discovery(understanding of the mechanisms of cytokine-independence, breakdown ofthe different SMPs) the evidencing of this mutation in a patient offersa test for the first time with which it is possible to confirmdiagnosis. From a medical diagnosis viewpoint, the search for themutation of JAK2 can be made on polynuclear neutrophils belonging to themalignant clone.

The invention also offers the determination of a specific treatment, ofkinase inhibiting type specific to the mutated protein, or gene therapy.

EXAMPLE 2 Detection of the JAK2 V617F Mutant for First IntentionDiagnosis of Erythrocytosis

2.1 Patients, Materials and Methods Comparison Between Sequencing andTwo Techniques of SNP Genotyping for the Detection of JAK2 V617F.

Patient Cells

119 samples of suspected MPD were analysed (i.e. erythrocytosis,thrombocytosis, hyperleukocytosis). 58 samples were taken forperspective analysis and 61 archive samples of bone marrow were analysedretrospectively.

The peripheral granulocytes were isolated using a density gradientmethod following the manufacturer's instructions (Eurobio, France).Mononuclear cells were isolated from the bone marrow using Ficolldensity gradient centrifugation. The genomic DNA was extracted followingstandard procedures. To determine the sensitivity of LightCycler® andTaqman® technologies, the DNA derived from a homozygous sample with theallele of minimum residual wild type was diluted in series in normalDNA.

Cell Lines

Serial solutions of DNA were used (1; 0.5, 0.1, 0.05, 0.01) from thehuman erythroleukaemia cell line (HEL) mutated homozygous fashion (JAK2V617F) in DNA of TF-1 cell line (non-mutated) as standard positivecontrols. The cells lines grew in MEM-alpha medium (Invitrogen) enrichedwith foetal calf serum.

Detection of the Mutation by Analysis of the Fusion Curve ofLightCycler® With FRET Hybridisation Probes.

Primers and probes were designed to amplify and hybridise to the targetsequence of exon 12 of JAK2. The position of the mutation site (1849G/T)was covered with a donor capture probe labelled with fluoresceine at 3′,and the adjacent acceptor anchor probe labelled with LightCycler® Red640 (LCRed640) at its 5′ end; its 3′ end was phosphorylated to avoidextension. Amplification and analysis of the fusion curve were performedon the LightCycler® instrument (Roche Diagnostics, Meylan, France). Thefinal reaction volume was 20 μl using 10 ng DNA, 14 μl LightCyclerFastStart DNA Master mixture, 3 mM MgCl₂, 0.2 μM primers, 0.075 μM ofeach probe. In brief, the samples were heated to 95° C. for 10 minutesand PCR amplification of 45 cycles (10 seconds at 95° C., 10 seconds at53° C., 15 seconds at 72° C.) was followed by a denaturing step at 95°C. for 10 seconds, two hybridisation steps at 63° C. and 45° C. for 30seconds each and a fusion curve located in the domain lying between 45and 70° C. (0.1° C./sec). Analysis on the LightCycler® programme wasperformed by plotting the curve of the fluorescence derivative withrespect to temperature [2(dF12/F11)/dT) versus T]. The mutated peaks andWT were observed at 57 and 63° C. respectively.

Detection of the Mutation by Specific Amplification of an Allele UsingTaqMan®

Two primers were designed to amplify a product of 92 by encompassing SNPat position 1849. Two fluorogenic MGB probes were designed withdifferent fluorescent colourings, one targeted towards the WT allele,and one targeted towards the mutated allele. Genotyping was conducted in96-well plates using the method based on Taqman® PCR. The final reactionvolume was 12 μl using 10 ng genomic DNA, 6.25 μl TaqMan® UniversalMaster Mix and 0.31 μl 40× Assays-on-Demand SNP Genotyping Assay Mix(Applied Biosystems). The plate was heated to 95° C. for 10 minutesfollowed by 40 denaturation cycles at 92° C. for 15 seconds andmatching/extension at 60° C. for 1 minute. Thermocycling was conductedon the 7500 Real Time PCR System (Applied Biosystems). Analysis was madeusing the SDS programme version 1.3. Genotyping of end point allelediscrimination was performed by visual inspection of a fluorescencecurve (Rn) derived from the WT probe against the Rn of the mutated JAK2generated from post-PCR fluorescence reading.

Patients With Erythrocytosis and Sample Collection

We evaluated 88 patients with hematocrit levels of more than 51%, at thetime of diagnosis, before any form of treatment, and we studied thepresence of WHO and PVSG criteria. The value of 51% was chosen for theupper end of the normal range for hematocrit level (Pearson T C et al,Polycythemia Vera Updated: Diagnosis, Pathobiology and Treatment.Hematology (AM. Soc. Hematol. Educ. Program.) 2000: to 68). Bone marrowcells were collected for clonogenic asays and excess cells werecollected for DNA extraction. Serum erythropoietin (Epo) was measured indifferent laboratories and it is therefore reported as being low whenbelow the lower value of the normal domain in each laboratory, normal orhigh. The peripheral granulocytes derived from the same patients werepurified as described previously, the blood samples of each time beingavailable. The samples of bone marrow and blood were collected afterreceiving informed consent.

EEC Assays

In vitro assays of erythroid Epo-response were all performed in the samelaboratory (Hotel Dieu, Paris) using a plasma-clot culture technique asdescribed previously (Casadevall N, Dupuy E, Molho-Sabatier P, TobelemG, Varet B, Mayeux P. Autoantibodies against erythropoietin in a patientwith pure red-cell aplasia. N. Engl. J. Med. 1996; 334: 630 to 633).

Statistical Analysis

Correlation of the markers was made using the Spearman rank correlationcoefficient (R).

2.2 Results

Feasibility and Sensitivity of Genotyping Techniques Based on PCR forDetection of the Mutation JAK2 V617F.

To assess the efficacy of sequencing, LightCycler® and Taqman®technologies for detection of the JAK2 V617F mutation, we searched itspresence in 119 samples taken from patients suspected of having a MPD,using the 3 techniques in parallel. The JAK2 V617F mutation wasefficiently detected in 83/119 samples, and 35 samples did not show themutation with any of the 3 techniques. In only one sample, sequencingfailed to detect the mutation revealed by the two technologiesLightCycler® and Taqman® thereby suggesting that the latter may be moresensitive.

To assess the sensitivity of the technique, we used two differentmethods: we tested serial dilutions of DNA of the HEL cell line withhomozygous mutation in the DNA of the non-mutated TF-1 cell line, andserial dilutions of the genomic DNA derived from a homozygous patientfor the mutation JAK2 V617F in normal DNA. Sequencing failed to detectthe mutated allele with 5% DNA of the HEL cell line diluted in the DNAof the TF-1 cell line, and with 10% of the DNA from the patient withhomozygous mutation diluted in normal DNA. The sensitivity of theLightCycler® and Taqman® techniques was equivalent, slightly better thansequencing, reaching 0.5 to 1% of the DNA from the HEL cell line dilutedin the DNA of the TF-1 cell line (FIGS. 6) and 2 to 4% of the DNA from apatient with homozygous mutation diluted in normal DNA.

Characteristics of Patients with Erythrocytosis at the Time of Diagnosis

The chief characteristics of 88 patients with hematocrit levels of morethan 51% at the time of diagnosis are summarized in Table I.

TABLE 1 Patient Characteristics WHO criterion PVSG criterion WHO andPVSG criteria Idiopathic Idiopathic Secondary Hct > 50% PVerythrocytosis PV erythrocytosis erythrocytosis but no AE n = 61 n = 11n = 45 n = 21 n = 5 n = 3 Sex ratio 38/23 11/0  28/17 18/3  4/1 3/0(male/female) Mean age 61 (23 to 92) 57 (24 to 81) 58 (23 to 92) 60 (53to 81) 65 (55 to 77) 48.6 (domain) mean Ht (%) ±  59 ± 4.6 54.6 ± 1.4459.2 ± 4.5  57.8 ± 4.2   55.8 ± 3.1 53.3 ± 0.8 σ Mean Hb 19.2 ± 1.3918.3 ± 0.34 19.3 ± 1.41 19 ± 1.0 18.9 ± 0.8 18.6 ± 0.5 (g/dL) ± σ MeanWBC 12.2 ± 4.4  7.0 ± 2.5 13.5 ± 4.9  8.2 ± 2.5   8.8 ± 1.9  6.6 ± 0.4(x/10⁹) ± σ Mean platelet 463 ± 148 212 ± 38  503 ± 149 245 ± 60.4 212 ±29 175 ± 19 count (x/10⁹) ± σ Splenomegalia 16/55  0/11 14/39  0/21 0/50/3 EEC presence 59/60  1/11 43/44 11/21 0/5 0/3 Low Epo level 39/47 2/827/33 10/17 0/3 1/1 Normal Epo  8/47 6/8  6/33  7/17 3/3 0/1 levelCytogenetic  7/32 0/3  6/23 0/7 nd 0/1 anomalies Positive 57/61  0/1143/45  8/21 0/5 0/3 JAK2V617F

88 patients with hematocrit levels of over 51% were diagnosed inaccordance with PVSG and WHO criteria into four groups: Vaquez disease(PV), idiopathic erythrocytosis, secondary erythrocytosis and “noabsolute erythrocytosis” (no AE) when measured red cell mass had notincreased. 8 patients could not have any definite diagnosis since someclinical data were not available. Hct: hematocrit; Hb: haemoglobin; WHC:white blood cells; EEC: endogenous erythroid colonies; Epo:erythropoietin; σ: standard deviation. The patients could be dividedinto 4 main groups in accordance with WHO criteria (Pierre R et al,editors, World Health Organization Classification of Tumours; Pathologyand Genetics of tumours of hematopoietic and lymphoid tissues. Lyon;IARC Press: 2001: 32 to 34) and PVSG criteria (Pearson T C, Messinezy M.The diagnostic criteria of polycythaemia rubra vera. Leuk Lymphoma 1996;22 Suppl 1:87 to 93): 61 and 45 patients with PV diagnosis, 5 withsecondary erythrocytosis, 11 and 21 with idiopathic erythrocytosis and 3with no absolute erythrocytosis (normal red cell mass). The clinicaldata were incomplete for 7 patients, accounting for the fact that PVdiagnosis could not be confirmed either with WHO criteria or with PVSGcriteria. On account of the difference between the A1 criteria of thetwo classifications, 6 patients who had no red cell mass measurementcould be classified in the WHO classification but not in the PVSGclassification. One patient showed both hypoxia and EEC formation,thereby making diagnosis difficult. Cytogenetic analysis was performedin 35 patients; among 32 PV patients (WHO criteria) 7 showed cytogeneticanomalies: 5 with trisomy 9, 1 with 7q- and 1 with additional materialon chromosome 18.

The presence of JAK2 V617F corresponds to PVSG and WHO criteria for PV

JAK2 V617F was present in 43/45 (96%) of patients diagnosed with PV inaccordance with PVSG criteria and in 57/61 (93%) of patients diagnosedusing WHO criteria (Table I). Nonetheless, 8/29 patients classified asnon-PV according to PVSG criteria showed the mutation, but none of the19 WHO non-PV patients; these 8 patients were considered IE with PVSGcriteria and PV with WHO criteria. None of the patients diagnosed withSE nor the patient with normal red cell mass (“no AE”) had the mutation.The presence or absence of JAK2 V617F therefore corresponds to positivePV diagnosis in 76/80 patients (95% R=0.879, p<0.0001) with WHOcriteria, and in 64/74 patients (86.5%, R=0.717, p<0.0001) with PVSGcriteria. In addition, since none of the patients diagnosed as non-PVaccording to WHO criteria showed the mutation, the detection of JAK2V617F has a 100% predictive value in the context of absoluteerythrocytosis.

Some authors (Mossuz P et al, Diagnostic value of serum erythropoietinlevel in patients with absolute erythrocytosis. Haematologica 2004; 89:1194 to 1198) consider the measurement of serum erythropoietin level asa first intention diagnostic test for patients with absoluteerythrocytosis, with a specificity of 97%, and a predictive value of97.8% for diagnosis of PV if the Epo level is below the, lower value ofthe normal range. In our study, the correlation between the Epo leveland PV diagnosis according to WHO and PVSG criteria was respectivelyobserved in 50/61 (82%, R=0.488, p=0.0002) and 39/56 (70%, R=0.358,p=0.0067) patients. We then compared the serum Epo level in the presenceor absence of V617F JAK2 and it was found that 52/68 patients (76%,R=0.416, p=0.0004) showed adequate correlation.

The presence of the JAK2 V617F mutation corresponds to the capacity forforming EECs.

Three different teams have shown that Epo-dependent cell linestransfected with JAK2 V617F were Epo-independent and Epo-hypersensitivefor their growth, thereby mimicking the independence andhypersensitivity of the erythroid progenitors described in PV.Therefore, we have put forward the hypothesis that patients carryingJAK2 V617F also showed EEC formation. Among the 20 patients witherythrocytosis with no EEC formation, one showed the mutation, raisingthe query of the positive predictive value of JAK2 V617F detection;however, even if this patient showed no EEC, he/she met the numerous WHOand PVSG positive criteria allowing the patient's classification as PVin both classifications. This patient should therefore be considered a“false-negative to EEC” rather than a “false-positive for JAK2”. Amongthe 67 patients who had EEC formation, 62 carried the JAK2 V617Fmutation, 5 patients not being mutated using detection-sensitivetechniques. Among these 5 patients, 4/5 and 2/5 could be classified inthe PV group according to WHO and PVSG criteria respectively. Overall,out of the 87 analysed patients, the presence or absence of the JAK2V617F mutation corresponded to the capacity or incapacity to form EECsin 81/87 patients (93.1%, R=0.824, p<0.0001).

The presence of the JAK2 V617F mutation in bone marrow mononuclear cells(BMMC) corresponds to its presence in the peripheral granulocytes.

To examine whether the use of granulocytes of peripheral blood to detectJAK2 V617F mutation at the time of diagnosis could avoid the assay ofbone marrow cells, we compared the results obtained by each of themethods: sequencing, LightCycler® and TaqMan®, in 50 patients (including35 PV, 8 SE and 8 suspected MPD) for whom both bone marrow samples andperipheral blood samples were available at the time of diagnosis. In allcases (34 mutated, 16 non-mutated) mutation was identically detected.

III—Conclusion

We therefore propose a new PV diagnosis datasheet in which the detectionof JAK2 V617F in the granulocytes using sensitive techniques is thefirst step in the diagnosis of eythrocytosis, except in the case ofobvious secondary erythrocytosis (FIG. 7). This approach has severaladvantages: it avoids having to conduct measurement of isotopic redblood cells, which is not always available and whose result is sometimessubject to debate. It can also avoid bone marrow procedure and EECassays which are time-consuming and are not well standardized. Itdrastically reduces the cost of positive PV diagnosis, since only thosepatients with hematocrit levels of over 51% and who are JAK2 V617Fnegative need to undergo all the investigations which are actuallycarried to characterize an erythrocytosis. Even if the detection aloneof JAK2 V617F in an erythrocytosis context will support PV diagnosis,performing a bone marrow biopsy may still be useful since it may revealsigns of myelofibrosis or the presence of blastic cells, therebyconfirming the leukaemic transformation of PV. Nonetheless, we feel thata bone marrow biopsy should be performed for optional study withcytogenetic analysis.

JAK2 V617F was also detected in 30% of ET, 50% of IMF and a few rarenon-characterized MPDs, thereby defining a new MPD group with differentclinical signs. The reasons for these differences remain unknown and itis still too early to group these diseases into a singlemyeloproliferative entity with a common physiopathological cause anddifferent phenotypes. Subsequent precise clinical studies wouldcharacterize more specifically the common signs between PV, ET, IMF andother rare MPDs carrying JAK2 V617F, especially in terms of absoluteerythrocytosis, Epo level, myelofibrosis and cytogenetic anomalies. Itis therefore contemplated to use the detection of JAK2 V617F as aninitial tool for the diagnosis of chronic hyperleukocytosis,thrombocytosis and erythrocytosis. The presence of JAK2 V617F will notonly allow a new definition of a MPD group, but it will also mostcertainly form the basis for developing specific targeted therapies.

EXAMPLE 3 siRNAs Specific to the V617F JAK2 Mutation Inhibit V617F JAK2But Not JAK2 WT

The siRNAs 1, 3 and 4 corresponding to sequences SEQ ID No 25 to 27inhibit the mutated protein V617F JAK2 expressed by the HEL line withoutinhibiting the wild-type JAK2 protein expressed by the K562 line. Theresults are shown FIGS. 8, 9 and 10.

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What is claimed is:
 1. A transgenic mouse whose genome comprises ahomozygous insertion of a variant human JAK2 transgene, the transgeneexpressing a JAK2 protein comprising the valine to phenylalaninemutation (V617F mutation) as set forth in SEQ ID NO: 1, said transgeneoperably linked to a regulatory sequence, wherein expression of thetransgene results in the transgenic mouse exhibiting a phenotype of PV(Polycythemia Vera) and myeloproliferative disorders induced by theV617F mutation.
 2. A method for identifying an agent as candidate forreducing or blocking the proliferation and spontaneous differentiationof erythroblasts of Vaquez polyglobulia, or for reducing cellproliferation associated with the presence of the V617F mutation,comprising the steps of: a) administering the agent to the transgenicmouse as defined in claim 1, and b) monitoring the transgenic mouse forreducing or blocking of the proliferation and spontaneousdifferentiation of erythroblasts of Vaquez polyglobulia, or for reducingcell proliferation associated with the presence of the V617F mutation.